In the sandwich technique, the undesirable bond between the composite resin and glass-ionomer cement (GIc) is one of the most important factors which lead to the failure of restoration. Total-etch and self-etch adhesives may improve the bond strength based on their pH. The purpose of this study was to evaluate the shear bond strength between the nanofilled composite resin and GIc using different adhesives.p< 0.05). In this experimental study, 40 specimens (6×6mm) in 4 groups (n=10) were prepared in acrylic mold. Each specimen contained conventional GI ChemFil Superior with a height of 3mm, bonded to Z350 composite resin with a height measured 3mm. In order to bond the composite to the GI, the following adhesives were used, respectively: A: mild Clearfil SE Bond self-etch (pH=2), B: intermediate OptiBond self-etch (pH=1.4), C: strong Adper Prompt L-Pop (pH=1), and D: Adper Single Bond 2 total-etch (pH=7.2). The shear bond strength was measured by using universal testing machine with a crosshead speed of 1mm/min. One-way ANOVA and Tukey’s test were used to analyze the data (p= 0.002), C (p< 0.001), and D (p< 0.001). Moreover, the shear bond strength of groups A and B (self-etch) was significantly different from group D  (p< 0.001); and C (self-etch) with D (p= 0.024). The shear bond strength in group A was significantly higher than group B ( The results of this study showed that applying the mild self-etch adhesive between the composite and the GIc results in stronger shear bond strength compared to intermediate and strong self-etch adhesives. Moreover, the self-etch adhesive increased the shear bond strength between composite resin and GIc more significantly than total-etch adhesive. Laminate technique or sandwich restoration is one of the methods used in dental composite restoration, in whichEtching the GIc is effective to obtain the favorable bond of composite. Using 35The bond strength between the conventional GIc and composite resin is due to the porosity in the etched surface of GIc. It has bIn order to reinforce the bond strength between the GIc and composite resin, surface treatment with self-etch system has been suggested. Since the self-etch system has less technique sensitivity, it can mostly meet the dentists’ need for using sandwich technique. The selfPrevious studies revealed the self-etch adhesive provided higher shear bond strength between the RMGIc and composite resin than other adhesives.-15 In a Apparently, the bond strength between GIc and composite resin considerably affects the clinical success of esthetic restorations. Hence, the present study was designed to evaluate the shear bond strength of nanofilled composite bonded to conventional GIc by using self-etch adhesives with different pHs and total-etch adhesive. In this experimental study, 40 specimens of 6×6mm were prepared in 4 groups (n=10) in acrylic mold (2.5×2.5 cm). First, a hole (3mm height×6mm diameter) was created at the top of this acrylic cylinder by using bur #14. This hole was filled with ChemFil Superior GIc  with a proportion of 2:2 in powder and liquid according to the manufacturer’s instruction. The excess of GIc was removed by celluloid strip and glass slap in order to put the GIc and the acrylic molding at the same level. It was accurately checked for each specimen. After 7 minutes of initial setting of the GIc, the adhesive resin was applied on the surface of GIc  . As reprIn group A, the mild self-etch adhesive was applied on the GIc surface according to the manufacturer’s instruction by using a microbrush. Then, it was mildly air-dried and light-cured for 10 seconds. Finally, the composite was applied on the GIc surface in two layers of 3-mm high and cured for 40 seconds. In groups B and C, the procedure was the same as what was done in group A, except that intermediate self-etch adhesive, and strong self-etch adhesive were used instead, respectively. In group D, after applying the mixed GIc and 7 minutes of rest for initial setting (according to the manufacturer’s instruction), the surface was covered by 37% phosphoric acid for 15 seconds, and was then rinsed. After thThe samples were all stored in distilled water for 24 hours at room temperature. The shear bond strength was evaluated by the universal testing machine  at a crosshead speed of 1 mm/min .The obtained data were analyzed by using SPSS software, version 20. One-way ANOVA and Tukey test were used to compare the mean shear bond strength among the groups. The significance level was set at 0.05. p= 0.002), C (p< 0.001) and D (p< 0.001). The maximum and minimum shear bond strength was obtained respectively in Group A with the mean of 7.77 MPa and group D  with the mean of 3.45 MPa.The mean±SD of shear bond strength of each group are presented in p= 0.002), and between group A and C (p< 0.001). A comparison of group B and C showed that the values obtained from them are significantly different (p= 0.002). Likewise, comparing groups A and B with group D (p< 0.001), and group C with D (p= 0.024) showed their shear bond strength were significantly different. Comparing the groups by Tukey’s test , a statiProper bond strength between resin composite and GIc is necessary for the success of sandwich technique. Applying self-etch adhesive over GIc creates a stronger bond of composite resin to GIc compared with total-etch adhesive. The totaOwing to the acidic monomer in its composition, the self-etch adhesive does not need the etching phase, rinsing and drying. Self-etcet al. who reported that the mild self-etch bonding provided higher shear bond strength.[Our study showed that the mild self-etch adhesive (Clearfil SE Bond) yields a higher shear bond strength between the ChemFil GIc and Z350 composite compared with the intermediate (OptiBond) and strong self-etch adhesive (Adper Prompt L-Pop). Similar results were achieved by Kandaswamy strength. This migstrength. Cations strength., 25 It sAdditionally, in our research, the strong self-etch adhesive system in group C (pH=1) had a lower shear bond strength compared with the other two groups of self-etch bonding (groups A and B). Some previous studies showed that using self-etch adhesive with a lower pH (1-0.8) created lower shear bond strength., 26-27Stet al. reported that the self-etch adhesive caused a stronger shear bond between the composite and RMGIc.[et al. on the same issue.[This research also found that self-etch adhesives improved the bond between the composite and conventional GIc compared with the total-etch adhesive. Arora nd RMGIc. Similar me issue. Another me issue.This mighme issue.Etching tme issue., 30 The On the other hand, self-etch adhesive has a lower viscosity compared with the total-etch adhesive. In a resPrevious studies on self-etch adhesives showed that this system bonds with the calcium in the structure of the teeth; thereforOverall, further studies are recommended to examine and evaluate the effect of different generations and the application of bonding with different pHs on the bond strength between the composite and light GI. Concerning the limitations of this study, it can be concluded that using the mild self-etch adhesive (Clearfil SE Bond) between the resin composite and GIc increases the shear bond strength compared with the strong (Adper Prompt L-Pop) and intermediate (OptiBond) self-etch adhesive. Moreover, using self-etch adhesive between the GIc and composite resin creates a higher shear bond strength compared with total-etch adhesive (Adper Single Bond 2).


To evaluate the advantages and efficacy of MRI-guided MWA, data including the accuracy of needle placement, scan duration, ablation time, total operation time, length of hospital stay, progression-free survival (PFS), and overall survival (OS) were collected and compared between the two groups. The mean number of machine scans required to adjust the needle position was 7.62 ± 1.69 (range 4–12) for the MRI-MWA group and 9.64 ± 2.14 (range 5–16) for the CT-MWA group (p < 0.001). The mean time for antenna placement was comparable between the MRI and CT groups . The microwave ablation time of the two groups was significantly different , while the overall procedure time was comparable . The overall complication rate in the MRI-MWA group was significantly lower than in the CT-MWA group . The median time to progression was longer in the MRI-MWA group than in the CT-MWA group . OS was comparable in both groups . This study provides hitherto-undocumented evidence of the clinical effects of MRI-guided MWA on patients with NSCLC and determines the relative safety and efficiency of MRI- and CT-guided MWA.Magnetic resonance-guided microwave ablation (MRI-guided MWA) is a new, minimally invasive ablation method for cancer. This study sought to analyze the clinical value of MRI-guided MWA in non-small cell lung cancer (NSCLC). We compared the precision, efficiency, and clinical efficacy of treatment in patients who underwent MRI-guided MWA or computed tomography (CT)-guided microwave ablation (CT-guided MWA). Propensity score matching was used on the prospective cohort (MRI-MWA group,  Percutaneous thermal ablation under imaging guidance is a minimally invasive technique used for the palliative treatment of nonoperative patients with non-small cell lung cancer (NSCLC) and pulmonary metastasis . As a heThis prospective study was approved by the Ethics Committee of Jiangsu Provincial Hospital, affiliated with Nanjing Medical University. All patients included in the study provided written informed consent.n = 45) with lung tumors treated with MRI-guided MWA combined with chemotherapy from February 2018 to August 2021 and a retrospective, observational cohort (n = 305) of consecutive patients who underwent CT-guided MWA combined with chemotherapy (CT-MWA group) at our hospital between January 2018 and June 2021. One-to-one propensity score matching (PSM) analysis was performed to remove confounding, selection, and information biases based on the following variables: gender , age (± 5 years), tumor size (± 4 mm), tumor location , histology, and TNM stage, yielding 39 patients in the MRI-MWA group and 39 patients in the CT-MWA group  was used to guide and to monitor the microwave ablation procedures with a 16-channel phased array coil. MRI-guided MWA was performed using an ECO-100A MWA system . Moreover, the cooled-shaft ceramic antenna (ECO-100CI8) was made of nonmagnetic ceramic material. The scanning parameters used are shown in All lung microwave ablations were performed using CT fluoroscopic guidance  with the following parameters: 5-mm collimation, 30 mAs, 120 kV, and 5-mm section thickness. The microwave system consisted of the ECO-100A MWA delivery system , and the cooled-shaft antenna (ECO-100AI13) was made of composite metal materials.The microwave ablation procedure was performed under aseptic conditions by three interventional radiologists with more than eight years of experience in thoracic intervention, a technician, and two experienced radiologists. Codeine phosphate tablets were used to relieve cough (30 mg per patient). Local anesthesia (1% lidocaine) was provided to ensure patients could tolerate the ablation procedure. Electrophysiological monitoring and pulse oximetry were conducted and recorded throughout the procedure. Power was generally 40–60 W, and the treatment duration was 8–16 min. At the end of treatment, patients were instructed to hold their breath. A 20-W power-track ablation was performed immediately to avoid tumor implantation metastasis. After therapy, MRI/CT scanning was performed to observe the incidence of complications and to evaluate treatment outcomes in order to determine whether additional ablation was needed.Multiple cod liver oil capsules were used as skin markers and were placed on the skin surface to determine the needle insertion site under MRI. The T1 and T2 sequences were combined to locate the puncture point and plan the preoperative puncture route. The whole-lung scan was performed on T2WI. Then, a breath-hold T1WI scan of the target region was performed. The microwave antenna has a low signal on the image. During the insertion process, horizontal, coronal, and sagittal MRI scanning was conducted during T1WI/TFE, providing a three-dimensional spatial relationship between the ablation needle and the tumor lesion, especially for irregular lesions. A vascular flow void on T2WI ensured that the puncture-ablation needle could effectively avoid important pulmonary vessels and prevent bleeding complications. T2WI/TSE, T1WI/TFE, and DWI sequences were scanned during MRI-guided ablation, and MWA dynamic intraoperative scans monitored changes in the MRI signals in the ablation foci. On T2WI, a low signal was found in the center of the ablated tumor, and the high signal area of the primary tumor disappeared and was surrounded by hyperemia and edema with a high signal, which was 5–10 mm beyond the primary tumor area, indicating successful ablation .CT scans were performed to determine the puncture point as well as the angle and depth of the needle. Scans were repeated until the ablation needle reached the center of the tumor. The effect of ablation was evaluated by observing the change in lesion density on CT imaging. After ablation, the tumor lesions showed extensive high density with features such as the “honeycomb sign” and the “bubble sign.”2 (injected for over 30 min). The drug was administered every three weeks on days 1 and 8. Cisplatin was administered at 75 mg/m2 every 3 weeks on day 1 (injected for over 60 min). The original protocol, when effective, was maintained for three cycles.Three courses of albumin-bound paclitaxel combined with carboplatin were used after microwave ablation. Albumin-bound paclitaxel was used at a dose of 125 mg/mA chest X-ray or CT scan was performed after 24–48 h to identify any complications. Conservative treatment was adopted if the patient had developed a small asymptomatic pneumothorax or pleural effusion without significant complications. Follow-up CT was performed at 1-, 3-, 6-, and 12-month intervals after the initial ablation session using a multidetector-row helical CT scanner. Contrast-enhanced chest CT images were used to evaluate outcomes.The primary outcomes were the accuracy of needle positioning and the efficiency and safety of ablation, as guided by the two imaging methods.The accuracy of ablation-needle puncture was defined as the targeting duration and number of scans required for ablation-needle placement.The ablation efficiency included the microwave ablation time (min) and overall procedure time (min). The overall procedure time was determined as the time between the patient’s arrival in the CT or MRI room and the time of their departure. In most cases, less than two ablation cycles were required, ranging from 4 to 8 min. Ablation cycle duration depended on lesion size and power output.Safety was assessed based on the presence of treatment-related complications, defined according to the Society of Interventional Radiology standard . SecondaImproved criteria from the Response Evaluation Criteria in Solid Tumors guidelines were used to evaluate the local tumor progression of patients at 3 years of follow-up .t-test or Wilcoxon rank-sum test. The χ2 test or Fisher’s exact test was used to compare categorical variables. The Kaplan–Meier method and log-rank test were applied to compare progression-free survival (PFS) and OS between groups. A p-value less than 0.05 was considered statistically significant.The variables were analyzed by SPSS 16.0 software. The means and standard deviations were determined for measurement data and were compared using the independent samples p > 0.05; Seventy-eight patients with lung cancer who underwent microwave ablation were included in this study. Patient demographics at baseline did not differ between the MRI and CT groups .There was no difference in the characteristics of the lesions, such as tumor size and depth. The mean number of scans required to adjust needle position was significantly less in the MRI group than in the CT group ). As shown in The median time to progression was significantly longer in the MRI-MWA group than in the CT-MWA group  (The median OS was 26.0 months [95% CI 25.022–26.978] in the MRI group and 23.0 months [95% CI 18.646–27.354] in the CT group ( = 0.18) .It is well-established that CT-guided microwave ablation provides a minimally invasive approach to eradicating tumors, especially for solid lung neoplasms . The advp < 0.001). Although the average single-scan duration for MRI was slightly longer than for CT, the average times for antenna placement were comparable for the MRI-MWA group and the CT group . The total procedure duration was comparable between both groups . Based on the accuracy of MRI-guided localization and the application of rapid sequence, the overall scanning duration and operation duration were comparable to the CT-MWA group. Taken together, the aforementioned findings suggest that the overall efficiency of MRI-guided microwave ablation is consistent with that of CT-guided microwave ablation and yields less damage due to a significant reduction in the number of needle adjustments.Our preliminary results show that MRI imaging yields superior accuracy, relative to CT, during ablation needle puncture. It is well-established that a fast sequence, such as the T1-TFE sequence, can distinguish anatomical structures for precise puncture. MRI can be used for arbitrary multidirectional imaging to ensure that the overall microwave antenna and the relationship between the microwave antenna and the lesion can be displayed during puncture, shortening the operation time and achieving a rapid, accurate puncture location. Moreover, the metal artifact produced by the ablation needle during CT can cover the ablation target. Importantly, magnetic resonance multisequence imaging and MR-functional imaging methods such as DWI, MRS, and PWI provide more accurate and abundant information than CT does for assessing the relationship between lung tumor lesions and surrounding tissues, cardiac great vessels, and the ablation boundary, making the puncture process simpler, safer, and more accurate . In our Indeed, the effect of ionizing radiation produced by CT scans, especially for tumor patients, should not be overstated . ImportaIn a previous study, we demonstrated a significant difference between the ablation power and the actual ablation power, and it was challenging to accurately control the ablation range . Excessi2 = 11. 237, p < 0.01), while no difference in OS was observed between the groups at 3-year follow-up .In the present study, we quantified the maximum diameter, including the ablation area, of isointense signal intensity on T1WI and of high signal intensity on T2WI images, without using contrast enhancement. These findings suggest that, compared with CT, MRI can provide a more accurate real-time evaluation of MWA. T2WI can be used to evaluate the effect of WMA. In the present study, the mean PFS was 11 months in the MRI-MWA group and 9 months in the CT-MWA group, and the difference was significant  after local tumor progression was not assessed in the 3-year follow-up, but this will be carried out during follow-up analysis. Additionally, the quality of life and other measures of patient experience of the treatment were not directly studied and should be considered in the future to truly reflect the value of MRI-guided microwave ablation for the treatment of NSCLC. Indeed, the follow-up period (3 years) was relatively short. A longer follow-up period may reveal differences in survival rates between the two groups.Overall, the results of our matched-cohort study suggest that MRI-guided percutaneous ablation has significant prospects for the treatment of lung tumors as a feasible, safe, effective, and minimally invasive approach and provides a satisfactory outcome.


A history of heavy drinking increases vulnerability to, and the severity of, Alzheimer’s disease (AD) and related dementias, with alcohol use disorder identified as the strongest modifiable risk factor for early-onset dementia. Heavy drinking has increased markedly in women over the past 10 years, particularly in mature adult women during the coronavirus (COVID-19) pandemic. This is concerning as women are more sensitive to many alcohol-related disease states, including AD and related dementias. Herein, we conducted two studies to determine if a 1-month period of binge drinking during mature adulthood  impairs spatial and working memory to a greater extent in female vs. male C57BL/6J (B6J) mice. The anxiogenic and cognitive-impairing effects of binge drinking were also compared between mature adult and old B6J mice (18 months of age) in a third study. Throughout, females consumed more alcohol than males, indicating that a sex difference in binge drinking persists into old age. Despite the sex difference in intake, we detected no consistent sex difference in our measures of alcohol withdrawal-induced anxiety during a behavioral test battery. Although mature adult females exhibited more cognitive deficits than males, the precise outcome exhibiting a female-selective effect varied across studies. Old mice drank lower amounts of alcohol than mature adult mice, yet their blood ethanol concentrations (BECs) were within error of the 80 mg/dl criterion for binge drinking, indicative of an age-related slowing of alcohol metabolism. As expected, 18-month-old controls exhibited more signs of cognitive impairment than their 6-month-old counterparts, and binge drinking history impaired the Morris water maze performance of mice of both ages. In contrast, binge drinking history impaired the radial arm maze performance of 6-month-old mice only, and the extent of the impairment was comparable to the behavior exhibited by the older mice. We conclude from our studies that: (1) both biological sex and the age of drinking onset are subject factors that impact voluntary alcohol consumption by mice into old age; (2) binge drinking during later life elicits a negative affective state that is relatively sex-independent; (3) binge drinking during both mature adulthood and old age impairs spatial learning and memory; (4) binge drinking during mature adulthood accelerates deficits in working memory; and (5) mature adult females tend to exhibit more alcohol-induced cognitive impairments than males. If relevant to humans, these findings suggest that binge-like drinking by older adult men and women induces a negative affective state and cognitive decline, but that mature adult women, in particular, may be more sensitive to both the immediate and persistent cognitive-impairing effects of heavy drinking. Alcohol use disorder (AUD) and dementia, including Alzheimer’s disease (AD) have a high rate of comorbidity  rodents has received even less experimental attention. However, there is consensus in the aging literature that the best animal model for both the normal and pathological aging process is the aging animal itself  a longer latency to locate the hidden platform(s) during the acquisition phases of both Morris water maze and radial arm maze testing; (2) less time spent in the quadrant of the Morris water maze during a memory probe test; (3) poorer reversal learning when the platform location was changed in the Morris water maze; (4) more reference and working memory errors during the acquisition of the radial arm maze; and (5) a greater reliance on non-working memory strategies to navigate the radial arm maze, as index by chaining behavior (see Section “Methods”). Third, we hypothesized that a history of binge drinking would augment the aforementioned signs of cognitive impairment in both male and female mice, but that the severity of the impairment would be more robust or comprehensive in female than male subjects.In all, three studies were conducted. The first experiment was a pilot study originally designed to assay the behavioral effects of binge drinking during middle-age , as well as their genetic and research experiential backgrounds. As summarized in Hnrnph1+/+ and +/– animals maintained at UCSB  mice from this cocaine study were housed under the reverse light-dark cycle for approximately 4.5 months until drinking commenced (see Some of the other mice employed in Experiment 1 were on a congenic B6J background and included wild-type (WT) mice from our colony of nced see .S11A7/S216AHomer2 mice, generated by the laboratory of Dr. Kimberly Huber (UT Southwestern). These mice arrived at UCSB on ~PND40–50, were housed in same-sex groups of 4, and quarantined for an additional 7 weeks, prior to housing in the main vivarium under the same reverse light-dark cycle as the other Experiment 1 mice. Following 2 weeks of acclimation to the reverse cycle, the Homer2 transgenic mice (~PND 120) were subjected to a cocaine-induced taste aversion experiment. For this, mice were individually housed in standard mouse cages with a wire top situated on a free-standing rack in the colony room at 2 h into the dark phase of the cycle (~1300 h). One hour later (~1400 h), mice were presented with a single sipper tube containing 20% sucrose (w/v) and allowed to drink for 1 h. This sucrose drinking procedure continued for 3 days. Immediately following the third sucrose drinking session, mice were injected, intraperitoneally, with 30 mg/kg cocaine and returned to their home cage. The next day, the sucrose drinking procedure was repeated to examine for taste aversion. Upon the completion of aversion testing, the mice were returned to their home cage and left undisturbed for ~3 months until alcohol drinking commenced. For the congenic B6J mice, only WT mice were included in this study to avoid potential interactions between the mutation and binge drinking  indicated that genetic/experimental background and/or age significantly adjusted the data for both binge drinking and the majority of our cognitive outcomes. Thus, to control for both variables, Experiment 2 was conducted using exclusively 6 month-old, male and female, mice on an isogenic B6J genetic background, and all mice were purchased from the Jackson Laboratory. Mice were housed in same-sex and same experimental condition groups of 3–4 under standard ventilated caging and reverse cycle housing conditions (lights off at 1100 h). The first cohort of B6J mice employed in Experiment 2 consisted of 24 male (12 Water and 12 DID) and 13 female B6J mice (7 Water and 6 DID). These mice arrived at our facility on PND22 and were housed under our reverse light-dark cycle until they commenced drinking procedures at 6 months of age—a time corresponding to late maturity in the B6J strain . Thus, Experiment 3 was conducted to directly examine for age differences in the effects of binge drinking on cognition. For Experiment 3, B6J mice arrived simultaneously at our facility at either 5 or 17 months of age and were housed for 1 month under our reverse light cycle prior to undergoing drinking procedures. The 6-month-old mice in Experiment 3 provided an unadulterated replicate of Experiment 2, while the 18-month-old mice served as a comparator for the severity of alcohol-induced cognitive impairment in the 6-month-old animals and to examine directly for age by sex interactions in the effects of binge drinking on our cognitive measures. At the start of Experiment 3, the samples sizes were 12 for each group, with the exception of the 18-month-old male Water control group, which had a sample size of 14 as two additional 18-month-old males arrived with the shipment and were placed in the control group to increase the statistical power of the study. One female 18-month-old water control was euthanized prior to behavioral testing for remaining underweight (~19 g) throughout the drinking phase of the study.ad libitum except during behavioral testing. All the cages were lined with sawdust bedding, nesting materials, and a plastic enrichment device in accordance with vivarium protocols. All experimental procedures were in compliance with The Guide for the Care and Use of Laboratory Animals  ethanol in tap water . Chloroform was used as the pre-solvents and the determination of ethanol from each sample was derived using the standard curve equation determined prior to analyses of the blood samples. A new standard curve was formulated for each cohort of blood samples to ensure maximal accuracy. After the ethanol peak area was determined, the peak area was used to determine the ethanol concentration and subsequently the percent of ethanol in the blood.On the 20th drinking day in Experiments 1 and 2 and on the 30th (last) drinking day in Experiment 3, submandibular blood samples were collected from the alcohol-drinking mice only, immediately after the 2 h alcohol-drinking period. The latter time-point in Experiment 3 merely reflected the availability of trained laboratory personnel to conduct the blood sampling procedures. Samples were stored at −20°C until processing. As conducted in recent reports  might reflect alcohol withdrawal-induced negative effect, the mice in Experiment 3 first underwent a behavioral test battery for negative affect prior to cognitive testing see . As in oTo test reactivity to a novel object as an index of neophobia-related anxiety  mice  were housed for only 1.5 months under a reverse light-dark cycle, while all of the male mice and the other fraction of female mice were housed under a reverse light-cycle since weaning (see Section “Subjects”). Thus, the data for Experiment 2 were also analyzed using an ANCOVA, adjusting for light-cycle as a covariate. The results of the statistical analyses indicated no adjustment of the means by the light-cycle covariate; thus, all data from Experiment 2, as well as the data from Experiment 3, are presented as the actual means ± SEMs. As there were no overt confounding variables in Experiment 3, the data were analyzed using multi-variate ANOVAs with the between-subjects factors of history and sex, with age (6 vs. 18 months old) also included as a between-subjects factor. Depending upon the paradigm, the within-subjects factors of day (for drinking), session (for maze acquisition), trial , or pre-pulse (for PPI) were also included in the analyses. Significant interactions were deconstructed along the relevant factors, followed by post-hoc tests for main effects (when multiple comparisons were involved) or t-tests (when <3 comparisons were involved). Statistical outliers were identified for each test using the _1.5_IQR rule and excluded from the analysis. Alpha was set at 0.05 for all analyses.As the mice employed in Experiment 1 were heterogeneous with respect to both their age at drinking/testing and their genetic/experimental background, the data for Experiment 1 were analyzed using an ANCOVA adjusting for both age and the genetic/experimental background as co-variates, with the between-subjects factors of History (Water vs. DID) and Sex. As one or both factors were found to significantly adjust the means for the vast majority of our dependent variables (see Section “Results”), all data from Experiment 1 are presented as adjusted means ± SEMs. A subset of the female mice in Experiment 2 (p’s < 0.05), or strong statistical trends towards a covariate effect . Thus, the data from Experiment 1 are presented as the adjusted means ± SEMs and all statistical results reflect the analysis of these adjusted means.Statistical analyses of the data by ANCOVA, adjusting for the difference in age of testing and the genetic/experimental background of the mice see  revealedn = 7) consumed more alcohol, on average, over the course of binge drinking than males  = 16.39, p = 0.001], with no sex difference detected for the time-course of drinking over the 30-day period (p’s > 0.50). Adjusting for age and genetic background, male (n = 11) and female (n = 7) mice consumed on average 4.63 ± 0.35 g/kg and 3.10 ± 0.28 g/kg alcohol, respectively, on the day of blood sampling and the intake on this day was significantly higher in females than in males . The adjusted data for the BECs attained on that day of blood sampling were quite variable, with the adjusted mean BEC of males exceeding the 80 mg/dl criterion for binge drinking, while that of females fell within error of that criterion (p = 0.39). These data indicate that mice aged 5–8 months engage in binge drinking under our 2-h DID procedures.When controlling for both age and genetic background, the female mice in Experiment 1  = 24.93, p < 0.0001; Sex × Drinking History, p = 0.56]. In fact, half of the 18 DID mice did not locate the flagged platform during the initial test, while only one of the 21 Water controls failed initially to locate the platform . The one Water mouse and seven of the nine DID mice were able to locate the platform on the 2nd trial, while the two remaining DID mice required a third trial before completing the task. These data indicate that a history of binge drinking during mature adulthood disrupts visually guided escape behavior. An analysis of the distance traveled during the flag test indicated that DID mice swam approximately five times a greater distance than Water mice during this test , the longer latency or inability to locate the visible platform does not reflect a motor impairment or a higher level of floating/passive coping behavior in the DID mice.After controlling for both age and genetic background, we detected a sex-independent increase in the latency of DID mice to locate a visually-cued platform during the flag test, compared to Water controls  . However, contrary to our findings for the Flag Test, DID mice were slightly, but significantly, quicker to locate the hidden platform during maze acquisition than Water controls (p’s > 0.13). However, we did detect a sex difference in learning as indicated by a significant Sex × Session interaction . As illustrated in F = 8.46, p = 0.006; for Sessions 2–4, all p’s > 0.08]. These data for the Morris water maze acquisition indicate that mature adult female mice exhibit initial deficits in spatial learning, but that binge drinking during mature adulthood does not impair spatial learning in this task.When controlling for age and genetic background, we detected an overall effect of prior binge drinking history on the latency of mature adult mice to locate the hidden platform across the 4 days of training . Thus, neither sex nor a history of binge drinking affects reversal learning in mature adult mice.Adjusting for age and background, we also detected no group differences in reversal learning in Experiment 1   with respect to the number of sessions required for mature adult mice to reach asymptotic performance in the radial arm maze. As illustrated in Adjusting for age and background, we detected a trend for a History × Sex interaction . As illustrated, this interaction appeared to reflect relatively poorer reference memory performance by male binge drinking mice very early during training (right), while female binge drinking mice tended to make more reference memory errors during the middle of the first week of training (right). We detected no group differences in the number of working correct memory errors committed during the first 7 days of maze training (p’s > 0.23). Although DID mice appeared to make more working memory incorrect errors in some trials than Water controls (p’s > 0.20). Likewise, we detected no group differences in the latency to complete the radial arm maze over the course of the first week of testing (p’s > 0.21). Taken together, these results indicate that when the age and background of the mice are taken into account, binge drinking during mature adulthood does not impact reference or working memory in the radial arm maze.When controlling for both age and background, we detected only a trend for a History × Session × Sex interaction for the number of reference memory errors committed across the first week of training in the radial arm maze , that reflected a relatively linear decline in chaining by males , but a linear increase in chaining by females . As illustrated in (left), males exhibited more chaining than females on session 2 , while females exhibited more chaining than males on session 7 . These data suggest that male mice attempt and then discard chaining as a strategy for maze navigation, while females come to employ this non-memory strategy as training progresses. Of relevance to our hypothesis concerning the effects of binge drinking on cognition, we also detected a significant History × Session interaction (right) , that reflected stable chaining behavior in Water mice , but a U-shaped function in DID animals , with DID mice exhibiting more chaining during session 2 , but less chaining during session 4  and session 5 , than Water controls. The U-shaped time-course of chaining behavior exhibited by DID mice suggests that they initially employ a non-memory strategy to navigate the maze, then discard this strategy relatively quickly (the next day) only to resume using this non-memory strategy later in training. Thus, mice with a history of binge drinking during mature adulthood are capable of adjusting their navigation strategy during radial arm maze training, indicative of intact cognitive flexibility, but a higher reliance on non-memory strategies for maze navigation.The failure to detect any binge drinking effect on our measures of radial arm maze performance prompted us to examine for differences in chaining behavior. Chaining refers to entries into adjacent arms and is a memory-independent navigation strategy that we have shown to be associated with anomalies in prefrontal cortex glutamate . Thus, all of the data from Experiment 2 are presented as unadjusted means ± SEMs and the statistical results reflect analyses of the unadjusted means.Statistical analyses of the data by ANCOVA, adjusting for the difference in time housed under a reverse light-cycle prior to the commencement of binge drinking procedures (1.5 months vs. 6 months), did not reveal any significant effect of this covariate for any of our measures  = 20.00, p < 0.0001] and a sex difference was detected in the time-course of alcohol intake  = 2.22, p < 0.0001]. Unexpectedly, the average alcohol intake of mature adult B6J mice of both sexes in Experiment 2 was approximately 1.5–2 g/kg lower than that reported previously by our group for 2 month-old adult B6J mice  = 2.90, p = 0.009]. Nevertheless, females exhibited a higher BEC than males  that was very close to the 80 mg/dl criterion for binge drinking  = 0.70, p = 0.04; for female DID vs. Water, t(20) = 2.15, p = 0.04; for male DID vs. Water, t(22) = 0.79, p = 0.44]. There was a statistical trend towards a significant History × Sex interaction for the distance traveled by the mice during the flag test that paralleled the latency data , arguing further that the poor performance of the DID females on the flag test does not reflect an inability to swim or more immobility/floating. However, distinct from Experiment 1, we detected no difference in the number of Water vs. DID mice that failed to locate the flagged platform when both sexes were considered . With the exception of 1 Water and 1 DID mouse, all of the B6J mice that required additional testing were able to locate the platform on the second trial, while the two remaining mice required a third trial before completing the task. While twice as many DID females failed to locate the platform on the first trial than female Water controls (6/10 DID vs. 3/12 Water), the proportion of female mice failing the task on the first attempt was also not statistically different . No group difference was detected for the proportion of Water vs. DID males failing the task on the first trial (1/12 DID vs. 2/12 Water). These findings from Experiment 2 provide our first evidence that mature adult females may be more sensitive to the cognitive-impairing effects of alcohol than males.As observed in Experiment 1 , a histoF = 3.63, p = 0.02], that was sex-independent . As in Experiment 1 (left), with post hoc comparisons failing to indicate specific Water-DID differences on any of the four training sessions (p’s > 0.09). Also akin to Experiment 1 (right) . As depicted in (right), the Sex × Session interaction reflected a longer latency of female mice to locate the hidden platform during Session 1 . These findings, derived from a homogenous cohort of 6-month-old mice, do not support any overt effect of binge drinking during mature adulthood on spatial learning in the Morris water maze.Replicating Experiment 1, we detected a significant effect of binge drinking on the acquisition of the hidden platform location in the Morris water maze  . This interaction reflected a significantly longer latency of DID females to enter the former platform location, relative to their Water controls (t(20) = 2.16, p = 0.046; males: p = 0.45]. Females also spent less time than males in the NE quadrant that formerly contained the platform , but this sex difference did not vary as a function of prior binge drinking history (p’s > 0.28). These data from the probe test indicate that 6-month-old B6J females exhibit signs of poorer spatial recall than age-matched males, and appear to be more sensitive than males to certain memory-impairing effects of binge alcohol.In contrast to the results of Experiment 1, a significant History × Sex interaction was detected for the latency to first enter the platform’s former location in the study of isogenic B6J mice . Thus, binge drinking during mature adulthood does not impair reversal learning in the Morris water maze.As observed in Experiment 1, no group differences in reversal learning were detected in the 6-month-old B6J mice of Experiment 2   and post-hoc tests confirmed that female DID mice required more days to acquire the maze than their Water controls , with no DID-Water difference detected in males (p = 0.38). These findings indicate that a history of binge drinking during mature adulthood impairs working memory performance selectively in female subjects.The pattern of group differences regarding the number of days required by 6-month-old B6J mice to reach asymptotic performance in the radial arm maze was very similar to that observed in Experiment 1 . HoweverF = 2.20, p = 0.05; History × Sex: F = 3.26, p = 0.08; other p’s > 0.30]. An inspection of the data in In contrast to Experiment 1, no significant group differences were detected regarding the number of reference memory errors committed across the first week of training , althougF = 2.00, p = 0.08; other p’s > 0.28], but we detected a significant History × Sex interaction for the mean number of working memory incorrect errors committed over the course of the first 7 days of radial arm maze training . As illustrated in t(20) = 2.73, p = 0.01; for males, p = 0.31]. Although DID females made more working memory incorrect errors than their Water counterparts, we detected no group differences in the latency of the 6-month-old mice to locate all four hidden platforms over the first week of training  = 9.70, p < 0.0001; other p’s > 0.28]. These data further indicate that binge drinking during mature adulthood does not produce a global disruption of radial arm maze performance but rather induces specific impairments in working memory to which females appear to be more sensitive.As reported for Experiment 1, we did not observe group differences in the number of working memory correct errors  , that reflected more chaining behavior in the DID vs. Water mice. However, in contrast to Experiment 1 (right), we did not detect any sex differences in chaining behavior in Experiment 2 . These data provide additional evidence that mice with a history of binge drinking during mature adulthood employ a non-memory strategy to successfully navigate a radial arm maze.Based on the chaining data from Experiment 1 , left, wriment 1 , right, F = 5 0.71, p = 0.02]. Although this sex difference appeared to be more pronounced in the 18-month-old mice  = 21.12, p < 0.0001; Age × Day, p = 0.21].On the day of blood sampling  = 23.72, p < 0.0001; Sex effect and interaction, p’s > 0.19]. Three statistical outliers were detected during the analyses of BECs that all exhibited very low BECs  and included one 6 month female, one 18 month male and one 18 month female and their data were excluded from the statistical analysis of the BEC data. The BECs attained indicated both an age- and sex-difference  = 5.54, p = 0.02; Age effect: F = 5.80, p = 0.02; interaction, p = 0.22] that reflected lower BECs in females vs. males and in the 6-month-old vs. 18-month-old mice. As depicted in The time courses of alcohol intake by female and male 6 and 18-month-old mice in Experiment 3 are presented in old mice , the intdrinking  . Curiously, we did detect an overall Age × History interaction , that reflected significantly lower startle, overall, in 18-month-old DID mice, relative to their Water controls (t(48) = 2.68, p = 0.01]. However, consistent with our prior studies of 2 month-old adult mice .Although B6J mice are well-characterized to exhibit age-related hearing loss  =F = 132.12, p < 0.0001]. Perhaps not surprising given their advanced age, 18-month-old mice exhibited weaker PPI at the 90 dB pre-pulse than their 6-month-old counterparts  = 9.55, p = 0.003; for 90 dB, t(96) = 2.77, p = 0.007; for 75 dB, p = 0.74]. Although binge drinking history reduced startle amplitude in the aged mice . These PPI data indicate that a recent binge drinking history does not impair sensorimotor gating in mature adult mice nor does it exacerbate the age-related decline in sensorimotor gating.Consistent with our published work in 2 month-old adult B6J mice , with both male and female DID mice making fewer light-side entries, relative to their water-drinking controls  = 24.45, p < 0.0001; Sex effect and interactions, p’s > 0.25). However, a significant three-way interaction was detected for the time spent in the light side . Deconstruction of the interaction along the age factor revealed no group differences in the 6-month-old mice , while a Sex × History interaction was detected in the 18-month-old mice . As illustrated in right, this interaction reflected, in part, a marked reduction in time spent in the light-side by 18-month-old DID females, relative to their Water controls , with a non-significant Water-DID difference observed in 18-month-old males . Deconstruction of the three-way interaction along the history factor also revealed a significant Age × Sex interaction in Water controls  that was not apparent in DID mice . As illustrated in (left vs. right), 18-month-old Water female mice spent considerably more time in the light-side than their 6-month-old counterparts , while no age-related difference was observed in Water control males . Taken together, these data extend to older mice the results of prior work in younger adult (2-month-old) mice  nor the number of open arm entries (p’s > 0.21). However, for both indices of anxiety-like behavior, we detected a main history effect , which reflected higher anxiety-like behavior in DID vs. Water mice. Although DID males exhibited the lowest level of open arm exploration of the groups tested  = 7.94, p = 0.006; for time in open arm: F = 3.53, p = 0.06, n.s.], with no interactions detected with the sex factor for either variable . Thus, in the elevated plus maze, a binge drinking history produces a negative effect state that is comparable both between male and female subjects, as well as between mature adult and old mice.Age did not impact the time spent in the open arm of the elevated plus maze  = 9.99, p = 0.002]. Deconstruction of this interaction along the Age factor revealed no group differences for the 6-month-old mice , but a male-selective reduction in close arm entries in 18-month-old DID mice (right) . Deconstruction of the three-way interaction along the history factor revealed a significant Age × Sex interaction in Water controls  that was not apparent in DID mice . In the case of closed arm entries, 18-month-old female and male water controls exhibited, respectively, less and more closed arm activity than their 6-month-old counterparts (left vs. right) . These latter data indicate that the effects of binge drinking on anxiety-like behavior in the elevated plus maze can be dissociated from effects on general activity, with respect to both sex and the age of the mice.The number of closed arm entries was also examined as a gross index of general motor activity in the elevated plus maze and analysis revealed a significant Sex × History × Age interaction , with a significant Water-DID difference confirmed in female [t(45) = 4.49 p < 0.0001], but not in male, mice . Also irrespective of age, a history of binge drinking reduced the number of novel object contacts . Although an inspection of F = 9.20, p = 0.003; other p’s > 0.19]. Thus, in the novel object encounter test, both 6 and 18-month-old females exhibit more signs of withdrawal-induced anxiety-like behavior than males.Irrespective of age, female DID mice exhibited the longest latency to first approach a novel object  . This overall binge drinking effect aligns with the results of the elevated plus maze, in which withdrawal-induced anxiety was both sex- and age-independent  = 18, 02, p < 0.0001]. As illustrated in right, the Age × Sex interaction reflected higher anxiety-like behavior in 18 month-old males vs. females , with no sex difference in marble-burying apparent in 6-month-old mice . These marble-burying data provide additional evidence that females exhibit an age-dependent reduction in anxiety-like behavior, but that binge drinking history induces an anxiety-like state, irrespective of the age or sex of mice.A history of binge drinking significantly increased the number of marbles buried, irrespective of either sex or age  . Deconstruction of the three-way interaction along the age factor revealed that 6-month-old females spent more time immobile than males (left) , and DID mice of both sexes spent less time immobile than Water controls . Comparable analyses of the data from the 18 month-old mice indicated a significant Sex × History interaction , that reflected less immobility in DID males vs. their water controls (right) . Deconstruction of the three-way interaction along the History factor indicated a significant Sex × Age interaction for Water controls  that reflected an age-dependent increase in the time spent immobile by male, but not by female, subjects . In contrast, no age-dependent change in immobility was detected in DID mice , with females spending more time immobile overall than males .Marked group differences were detected with respect to the time spent immobile in the forced swim test . Although inspection of p’s > 0.10). That being said, we did detect a significant Sex × Age interaction  that reflected more immobility in 6-month-old females than males (left) , with no sex difference apparent in 18-month-old mice . Thus, as reported for younger adult (2-month-old) mice  = 4.18, p = 0.04; Sex effect: F = 5.93, p = 0.02]. Although it appeared that a history of binge drinking increased the time to locate the flagged platform more so in 6-month-old animals (left) than in 18-month-old animals (right), the results of the statistical analyses did not support this observation . Thus, the magnitude of the alcohol-induced impairment in visually-cued navigation did not vary significantly as a function of aging. An inspection of t(21) = 0.58, p = 0.57; for males, t(24) = 0.36, p = 0.73]. These findings replicate the results of the prior two studies, indicating that binge drinking impairs the performance of mature adult mice on a visually-cued spatial navigation task, extend these findings to old mice and provide evidence that binge drinking during mature adulthood advances the onset of this particular cognitive impairment in both male and female mice.Not surprisingly, 18-month-old mice took longer to locate the flagged platform than did the 6-month-old mice . Deconstruction this is three-way interaction along the Sex factor revealed a significant Drinking × Session interaction for female mice , that reflected a longer latency for DID vs. Water females to locate the hidden platform on Session 1 of training (t(45) = 5.66, p < 0.0001; for other sessions, t’s < 0.80, p’s > 0.43]. In contrast, binge drinking history did not affect learning in males  = 28.84, p < 0.0001; other p’s > 0.14]. The Age × History × Session interaction was shy of statistical significance (p = 0.09), indicating that the drinking-induced impairment in spatial learning was not significantly different between 6 and 18-month-old mice. However, we did detect a significant Age × Sex × Session interaction  for spatial learning that reflected poorer spatial learning by female vs. male 6-month-old mice (left) , but no sex difference in the learning for 18-month-old animals (right) .Analyses of the time taken to locate the hidden platform in the Morris water mazeindicated that sex significantly influenced the effect of binge drinking on learning , indicative of an age-related impairment in spatial recall. However, irrespective of age, DID mice exhibited a longer latency to first enter the former platform location . Although inspection of p = 0.06), owing likely to the larger variability in the data for females of both ages. To test the hypothesis that a binge drinking history elicited a comparable impairment in this measure of spatial recall as that produced by normal aging, we conducted planned comparisons between the 6-month-old DID mice and the 18-month-old Water controls. As suggested by (left vs. right), this comparison revealed no statistically significant difference , indicating that binge drinking during mature adulthood accelerates impairments in the spatial recall. However, in line with Experiment 2, we detected no group differences for the total time spent in the former platform quadrant (p’s > 0.26). Taken all together, the results from our two studies of B6J mice argue that the latency to the platform location is more sensitive to the recall-impairing effects of alcohol than the more classic dependent variable of time spent in the former platform quadrant.Irrespective of sex, 18-month-old mice exhibited a longer latency to first enter the former platform location than younger mice  = 20.26, p < 0.0001], that reflected a longer and shorter average latency, respectively, for DID vs. Water female and male mice to find the relocated platform over the four training trials (t(45) = 3.63, p = 0.001; for males, t(48) = 2.66, p = 0.01]. Thus, regardless of the age of drinking onset, the female mice in this study were more sensitive overall to an alcohol-induced impairment in reversal learning. This being said, we also detected a significant Age × History × Trial interaction . As illustrated in p’s > 0.06; Age × Trial ANOVAs for Water vs. DID mice: p’s > 0.14; Age × History univariate ANOVAs conducted separately on trial 1, 2, 3 or 4: p’s > 0.06]. Taken together, the data in post hoc analyses, indicate that the alcohol-induced impairment in reversal learning is age-independent.An analysis of the time taken to find the relocated platform during the reversal test indicated a significant overall Sex × History interaction , the deconstruction of the interaction along the Sex factor  and post hoc comparisons between Water and DID females at each age group . In fact, the magnitude of the binge drinking effect in the younger, 6-month-old, female mice obscured the detection of an age-dependent increase in the time taken to acquire the radial arm maze in female subjects in this study , which was readily apparent in male mice . These findings with respect to the number of days required to reach the acquisition criterion in the radial arm maze replicate the results of Experiment 2 and further the observation that a history of binge drinking during mature adulthood produces a female-selective impairment in working memory performance, with little evidence for an alcohol-induced perturbation in maze learning in more aged mice.In Experiment 3, a prior history of binge drinking appeared to selectively increase the number of days required by 6-month-old female mice to reach asymptotic performance in the radial arm maze . This obleft vs. right)  and binge drinking differentially modified the time-course of reference memory errors in 6- vs. 18-month-old mice . Deconstruction of this three-way interaction along the session factor indicated that 18-month-old Water controls committed more reference memory errors on session 2 than their 6-month-old counterparts , with no age-related difference in reference errors detected in DID mice (p = 0.85). On session 3, both 6 and 18-month-old DID mice committed more reference memory errors than their Water controls . No group differences were detected during sessions 4, 5 or 7 , while several group differences were noted on session 6 , including a greater number of reference errors by 18 vs. 6-month-old Water controls (p = 0.03), and by 6-month-old DID mice vs. their Water controls (p = 0.02). Addition, as well as fewer reference memory errors by 18-month-old DID mice vs. their Water controls (p = 0.005). Taken together, these data for reference memory errors provide some evidence that binge drinking history can influence spatial reference memory also in the radial arm maze, with 6-month-old mice exhibiting more consistent signs of impairment than aged mice.Irrespective of sex, 18-month-old mice made significantly more reference memory errors during the first week of radial arm maze training than their 6-month-old counterparts  = 5.36, p = 0.02]. Despite this, we detected no significant interactions with the Sex factor on this measure . Inspection of p = 0.75). We did detect a significant Age × History interaction , that reflected more working memory correct errors in 6-month-old DID mice vs. their water controls , with no binge drinking effect apparent in 18-month-old mice . Planned comparisons conducted between the number of working memory correct errors committed by 6-month-old DID and 18-month-old Water controls confirmed that the alcohol-induced working memory impairment exhibited by the younger mice was comparable to that of aged animals (left vs. right) .Analysis of the number of working memory correct errors committed by the mice in Experiment 3 indicated more errors overall in females vs. male subjects . Also irrespective of sex, binge drinking history increased the average number of working memory errors committed by 6-month-old mice during the first week of training (left), with no binge drinking effect detected in 18-month-old animals (right) . As observed for working memory correct errors (left vs. right) , providing further evidence that a history of binge drinking during mature adulthood accelerates the onset of working memory impairments.An examination of the time-course of the number of working memory incorrect errors committed during the first week of radial arm maze training indicated that, irrespective of sex and drinking history, 18-month-old mice committed more errors than their 6-month-old counterparts, notably on sessions 2 and 6 of training  . Although 18-month-old mice required more time to navigate the radial arm maze than the younger mice, overall  = 44.56, p < 0.0001], 6-month-old DID mice took longer to navigate the maze, relative to their Water controls, while the opposite pattern of effect was observed in 18-month-old mice . Although sex did not significantly influence the average amount of time required by DID and Water mice to complete the radial arm maze, we detected two significant 3-way interactions in which sex modified the time-course of learning over the first week of radial arm maze training. Deconstruction of the significant Sex × History × Session interaction  along the sex factor did not detect any binge drinking effect in female subjects (left) , while a significant interaction was detected for males (right) . Male DID mice spent more and less time, respectively, than their Water controls to locate the platforms on sessions 3 and 5 of training . Deconstruction of the significant Age × Sex × Session interaction  along the Sex factor detected a main age effect for female subjects (left) , that reflected a longer latency to complete the maze in 18 vs. 6-month-old mice. For males, a significant Age × Session interaction was detected  that reflected a longer latency of 18-month-old males to complete the maze on sessions 2–6 . These later findings reiterate the results presented in In contrast to the results of Experiment 2, an analysis of the amount of time taken for 6 and 18-month-old mice to complete the radial arm maze during the first week of training revealed an overall Age × Drinking History interaction . Deconstruction of the interaction along the Age factor indicated a History × Sex interaction for both 6 and 18-month-old mice . For the 6-month-old mice (left), the interaction reflected more chaining overall in DID females vs. their Water controls . In contrast, for the 18-month-old mice (right), the interaction reflected non-significant trends for more chaining by females, but less chaining my males . We also detected a significant Age × Sex interaction for chaining , that reflected more chaining by 18 vs. 6-month-old males  , with a non-significant trend towards the opposite effect detected in females , owing likely to the large binge drinking effect on chaining exhibited by 6-month-old females  . Although we detected a female- and age-selective effect of binge drinking on the average amount of chaining behavior exhibited by the mice in Experiment 3  = 2.36, p = 0.04; other p’s > 0.12]. As illustrated in F = 8.86, p = 0.005], that of DID mice followed a quadratic function , with DID mice exhibiting significantly more chaining behavior than Water mice on the 7th training session . These data provide additional evidence that mice with a history of binge drinking employ a non-memory navigational strategy to complete the radial arm maze.An analysis of chaining behavior in Experiment 3 revealed a significant Age × History × Sex interaction [old mice , left, told mice , right, riment 3 , neitherAUDs are identified as the strongest modifiable risk factor for dementia onset . Acknowledging the obvious potential confounds associated with studying a heterogeneous group of mice in Experiment 1, we rationalized that if the sizes of any sex and/or alcohol effects were sufficiently large, they should be detectable, or at the very least, indicative of data trends worthy of follow-up investigation. Indeed, when adjusting for the co-variates of age and background, the results from Experiment 1 were largely negative , binge drinking induces a robust negative affective state that is apparent at 24 h into alcohol withdrawal . As all of the mice in the present study were tested over the first 5–6 h of the 12-h dark phase, it is not likely that the hyperactivity of the 18-month-old males expressed in the elevated plus maze reflects “sundowning syndrome”. Taken all together, the results of the present study and those of prior behavioral screens of older mice  induces anomalies in hippocampal microglia that are not observed in adolescent binge drinking mice (Grifasi et al., The 18-month-old mice in the present study consumed less alcohol than their younger counterparts but achieved a BEC within error of the 80 mg/dl criterion for binge drinking (National Institute on Alcohol Abuse and Alcoholism, To our surprise, we failed to detect any alcohol-induced impairment in the radial arm maze performance of 18-month-old mice. In fact, the only alcohol effect detected for 18-month-old mice was a quickening of the time taken to locate all 4 hidden platforms during maze training —an effecIndeed, it is worth noting that in none of our three experiments did we detect any effect of binge drinking or age on reversal learning in the Morris water maze . ReversaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the University of California Santa Barbara.KS and CJ: conceptualization and supervision. KS: formal analysis, writing—original draft preparation, visualization, and project administration. KS, EVD, JM, NO, AK, LM, IK, JH, JT-G, ER, and CJ: investigation, writing—review and editing. KS, CJ, IK, LM, and NO: funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The increase in specificity was mainly driven by the ability of LC-OCT to differentiate BCCs from other diagnoses. In conclusion, our real-life study showed that LC-OCT can play an important role in helping the noninvasive diagnosis of malignant skin neoplasms and especially of BCCs. LC-OCT could be positioned after the dermoscopic examination, to spare useless biopsy of benign lesions without decreasing sensitivity.Line-field confocal optical coherence tomography (LC-OCT) is a new, noninvasive imaging technique for the diagnosis of skin cancers. A total of 243 benign (54%) and malignant (46%) skin lesions were consecutively enrolled from 27 August 2020, to 6 October 2021 at the Dermatology Department of the University Hospital of Siena, Italy. Dermoscopic- and LC-OCT-based diagnoses were given by an expert dermatologist and compared with the ground truth. Considering all types of malignant skin tumours , 22 squamous cell carcinomas, and 10 melanomas), a statistically significant increase ( Line-field confocal optical coherence tomography (LC-OCT) is a new, noninvasive skin imaging technique that combines the advantages of optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) in terms of spatial resolution, penetration, and image orientation, overcoming their respective limits ,2,3,4,5.Recently, LC-OCT  has beenMany descriptive studies have shown the relevance of this imaging device for the diagnosis of cutaneous tumours, and our study aimed to evaluate the sensitivity and specificity of LC-OCT compared to dermoscopy for the diagnosis of skin tumours in a real-life setting in a third-level dermatology department. Prospective observational, monocentric study.Patients were enrolled from the 27 August 2020 to the 6 October 2021 at the Dermatology Department of the University Hospital of Siena, Italy, from the melanoma prevention outpatient ambulatory. The study was conducted according to the criteria set by the Declaration of Helsinki. All data were deidentified before use. We enrolled consecutive patients with cutaneous lesions of clinical and/or dermoscopic uncertain diagnosis of possible malignant skin tumours that needed to be removed or followed up according to a skin imaging expert dermatologist (E.C.) and that had LC-OCT examination.Dermoscopy was performed both with a hand-held 10× dermoscope  and a 20× videodermoscope . LC-OCT  was performed in horizontal and vertical scans and 3D mode, and a video was acquired on the operator’s judgment. Following manufacturer recommendations, lesions in the patients’ periocular region were excluded from LC-OCT examination. Dermoscopic and LC-OCT diagnoses were given by an expert in skin imaging (E.C.) during the imaging examination of the lesions and were registered on the LC-OCT device. Concerning LC-OCT, BCC was diagnosed by the presence of tumour lobules , SCC by The sensitivity and specificity of each technique (LC-OCT and dermoscopy) for the diagnosis of BCC, SCC/Bowen disease , AK/SCC/Bowen disease  group and malignant tumour were calculated with their exact 95% confidence interval (CI) by using the histopathological diagnosis obtained from an incisional or excisional biopsy as the gold standard; a distinct analysis was also performed, including the lesions that had a final diagnosis based on a follow up of at least one year without a histological examination. p values < 0.05 were considered statistically significant. All statistical analyses were conducted by using R . The sensitivity and specificity of LC-OCT and dermoscopy for the diagnosis of the different skin tumours were compared by using the proportion test. We included 196 patients  with 243 lesions; 226 lesions were histopathologically confirmed  and 17 lp = 0.015) for BCC diagnosis than dermoscopy , and no statistically significant difference in sensitivity  for SCC/Bowen disease diagnosis than dermoscopy , which did not reach a statistically significant difference. Concerning specificity, no statistically significant difference was found  for dermoscopy and 0.95 (CI 0.82–0.99) for LC-OCT, specificity of 0.96 (CI 0.92–0.98) for dermoscopy and 0.97 (CI 0.93–0.99) for LC-OCT ). Howevep = 0.013) for a malignant tumour, whereas the sensitivity was similar with the two imaging techniques (0.95 CI 0.89–0.98 for dermoscopy and 0.95 CI 0.90–0.99 for LC-OCT). The group of malignant tumours included both skin carcinomas and melanomas.Considering only the cases with a histologic diagnosis of malignancy, we observed a significant increase of specificity from 0.73 (CI 0.64–0.81) with dermoscopy to 0.87 (CI 0.79–0.93) with LC-OCT . Sensitivity was similar for LC-OCT  and dermoscopy , the specificity for BCC diagnosis increased from 0.79 (CI 0.72–0.85) for dermoscopy to 0.96 (CI 0.91–0.98) for LC-OCT (p < 0.001) for LC-OCT. Sensitivity was similar: 0.95 (CI 0.90–0.99) for LC-OCT and 0.95 (CI 0.89–0.98) for dermoscopy , the specificity for malignancy increased respectively from 0.64 (CI 0.55–0.72) of dermoscopy to 0.89 . The sensitivity was similar with the two methods (0.96 CI 0.89–0.99 for dermoscopy with three FN cases and 0.97 CI 0.91–1.00 for LC-OCT with two FN cases). Our study showed that LC-OCT can increase specificity for the noninvasive diagnosis of skin cancers compared to dermoscopy. Considering histopathology as a gold standard and analyzing only the cases with histological diagnosis , we founp < 0.001), whereas with regard to sensitivity we did not find any statistically significant difference between dermoscopy (0.96 CI 0.89–0.99) and LC-OCT (0.97 CI 0.91–1.00). Similar sensitivity results probably reflect the current use of LC-OCT as a secondary-level technique on skin lesions that are already identified as suspicious by dermoscopic examination. In most cases, LC-OCT easily confirms the dermoscopic diagnosis of a malignant tumour [The same analysis including the cases that were diagnosed based on a follow-up of at least one year and that lacked histopathological examination obtained similar results. We found an increase in the specificity for the diagnosis of BCC from 0.79 (CI 0.72–0.85) for dermoscopy to 0.96 (CI 0.91–0.98) for LC-OCT  BCCs in our series. 13 FP cases of BCC at dermoscopy were correctly diagnosed with LC-OCT . These dThere were only seven FP cases at LC-OCT, and three of them corresponded to benign skin tumours that can share histopathological similarities with BCC: a sebaceoma, a trichoblastoma, and a trichilemmoma. Sebaceoma  is charaIn the literature, we could find only one prospective study on the diagnostic accuracy of LC-OCT for skin tumours, and it consists of similar real-life research on equivocal lesions. It showed that LC-OCT can significantly increase diagnostic confidence after dermoscopy and avoid potentially unnecessary biopsies . HoweverThe acquisition and interpretation of the LC-OCT images are operator-dependent and different results may be related to different investigator expertise and different lesion selection . Moreover, Gust et al. found that in 70% of the lesions, the LC-OCT diagnostic was provided with high confidence in comparison with dermoscopy which only provided high confidence in 48% of the lesions . In thisp = 0.013). However, we did not detect any statistically significant difference in sensitivity between the two methods (0.95 IC 0.89–0.98 for dermoscopy and 0.95 IC 0.90–0.99 for LC-OCT), similar to the diagnosis of BCCs. The same analysis including the 17 FP cases of malignity at dermoscopy in which the diagnosis of malignant neoplasm was excluded with LC-OCT and surgery was not performed  while regarding the sensitivity we did not find any statistically significant difference between dermoscopy (0.96 CI 0.89–0.99) and LC-OCT (0.97 CI 0.91–1.00). These data on malignant tumours were mainly driven by BCCs and SCCs because melanomas were few. The increase in specificity with LC-OCT for the diagnosis of malignant skin tumours was mainly determined by the increase in specificity for the diagnosis of BCC. Although LC-OCT seems to play a possible role also for melanocytic tumours [Regarding the diagnosis of malignancy, considering histopathology as a gold standard and analyzing only cases with histological diagnosis, a significant increase in specificity  was obseour, see ) obtaine tumours , to date tumours .Our real-life study confirmed that dermoscopy can select lesions at risk of being malignant skin tumours (very sensitive tool).LC-OCT could be positioned in a second line to rule out malignancy to spare useless biopsy without decreasing sensitivity (very specific tool).LC-OCT can help in the identification of BCC with only 10 diagnostic errors in our entire database covering more than one year.LC-OCT seems to also be promising for keratinocyte tumours  by increasing the specificity and reducing FP cases compared to dermoscopy.Further studies should be performed to confirm our data and investigate the possible role of LC-OCT for the different malignant skin tumours.


CCL5+ B-cells in HS and the presence of SESN1+ B cells in HS with hepatocellular carcinoma (HS-HCC). In cell-cell interaction analysis, CD74-COPA and CD94-HLA-E in HS were found to be up-regulated. We further explored HS-specifically enriched genes  and found that FKBP5 was highly expressed in HCC-HS, leading to immunosuppression. Taken together, this research provides new insights into the genetic characteristics of HS via comprehensive single-cell transcriptome analysis.Hypersplenism (HS) is a concomitant symptom of liver or blood disease. Not only does the treatment of HS face challenges, but the transcriptome of individual cells is also unknown. Here, the transcriptional profiles of 43,037 cells from four HS tissues and one control tissue were generated by the single-cell RNA sequencing and nine major cell types, including T-cells, B-cells, NK cells, hematopoietic stem cells, neutrophil cells, mast cells, endothelial cells, erythrocytes, and dendritic cells were identified. Strikingly, the main features were the lack of  Hypersplenism (HS), as a clinical syndrome, is mainly characterized by splenomegaly and a significant reduction in one or more blood cells , 2. The Recently, single-cell RNA sequencing (scRNA-seq) has offered new possibilities to address biological and medical questions –19. UnliNo investigation using scRNA-seq on HS has yet been reported. The cell types and specific genes in HS are unknown, making it necessary to understand the genetic characteristics of HS and find possible treatments. Hence, in this study, five spleen samples were collected and scRNA-seq was performed through the 10× Genomics sequencing platform. The cell subtypes in each sample and the specific genes for each enriched cell group were investigated. Additionally, the evolutionary trajectory among different types of HS was also constructed to expound the relationship among these HS.Spleen tissues from HS patients with hepatitis B virus , portal hypertension , hepatitis C virus , and hepatocellular carcinoma , and one patient with a normal spleen (PN021) were used Table 1.All samples were digested to a single-cell suspension and studied by 10× Genomics scRNA-seq techniques S1D. FurIn HS samples, T-cells and B-cells were the main cell subsets (accounting for 86.8%), and significant differences in T-cells and B-cells were found among different types of HS (+ T-cells (44.33%), and 17 clusters belonged to CD8+ T-cells (55.67%) , CD4+ T helper cells , CD4+ Tregs cells , CD8+ T helper cells , CD8+ Naïve cells , CD8+ Cytotoxic cells , CD8+ MAIT cells   sample but was not enriched in PH026 (HS-PHT) samples. We also enriched some genes related to immune and diseases, such as the specific expression of TNFSF8 in T-cells of HS samples, FKBP5 specific expressed in T cells and other cells of HS-HCV. In addition, some enriched genes in T cells were also found in HS. DIP2A and SH2D2A and both were highly expressed in the CD8+ cytotoxic cells. In addition, DIP2A was associated with abnormal brain development and brain diseases  leading to their gene silencing . The T/B-cells enriched in HS exhibited abnormalities in the developmental trajectory, among which PH037 was the most obvious.via CD74. In addition, NK cells simultaneously play a regulatory role on T-cells and B-cells through CD94-HLA-E. Previous studies have found that NK cells activate T/B-cells earlier and exert a regulatory effect thereon . These abnormal B-cell subclusters regulate a variety of cells (including T cells) through CD74, which in turn leads to a systemic immune imbalance. After splenectomy, abnormal B-cells are removed, and normal immunity is restored. Cell communication analysis also provides potential drug targets for treating the disease.Subsequently, the interactions of various cells in HS were elucidated through cell interaction and ligand-receptor analyses. The cell-cell interaction analysis found that the main interaction cells in HS are epithelial cells and HSC. Peripheral blood cell reduction is one of the most common complications of HS and the spleen can regulate the function of HSC by regulating EGF signals through epithelial cells . However thereon . CombiniCCL5+ B cell deficiency). Many immune-related genes were specifically expressed in HS, especially FKBP5 and RPS4Y1. The significantly enriched genes and pathways provide new insights into the genetic and molecular characteristics of HS, which is critical to develop more potential treatments for HS.In summary, we have innovatively drawn a whole-cell map of HS. These results implied that HS is an immune disorder with a variety of cell imbalances caused by B cell  and a patient with normal spleen (group: PN) were selected. The splenomegaly of PH group patients was caused by different reasons, including hepatitis B virus infection , portal hypertension , hepatitis C virus infection , and hepatocellular carcinoma . The patient with a normal spleen was used as control (PN027), whose spleen was ruptured due to trauma.This study was approved by the Ethics Committee of Shanxi Bethune Hospital, and complied with all relevant ethical regulations (SBQLL-2020-038). Each participants signed an informed consent proforma.Samples were freshly collected from patients during surgical resection. Firstly, the spleen in the abdominal cavity was removed from its blood supply and quickly excised by surgical instruments. To maintain cell viability, each specimen was immediately immersed in culture medium and stored in separate 50 ml centrifuge tubes with 10% fetal bovine serum (FBS). Then, about 0.1-1 g spleen tissue was transferred into a 100-mm Petri dish and was cut into 1-2 mm tissue blocks using sterilized scissors and tweezers. The tissue blocks were transferred to a 10 ml centrifuge tube and were washed two times by adding PBS solution, centrifuging at 300g for 5 minutes, and removing the supernatant. Thereafter, the tissue blocks were transferred to cryopreservation tube and 1 ml of cryopreservation fluid was added . After standing at room temperature for ten minutes, the samples were stored at -80 °C for further investigation. All laboratory work in this study was conducted in a biological safety cabinet to prevent contamination.To maintain cell structure, spleen tissue was dissociated into single-cell suspensions by enzymatic degradation of the extracellular matrix. Firstly, the tissue was washed by using ice-cold PBS buffer and soaked in 30 mM EDTA solution for 20 mins on ice to inhibit nuclease activity and reduce the stability of the cell membrane. The tissue was then washed again using PBS buffer, and 2 mg/ml collagenase IV  was used to dissociate tissue into single cells at 37°C for 20 minutes. To maintain cell viability, the single-cell suspension was put at ice and 10% FBS solution added. After dissociation, the suspension was filtered through a 40-um cell strainer  to remove large particles.Single-cell barcoding and scRNA-seq library preparation were performed based on 10X Genomics single-cell RNA sequencing platform . Before barcoding, the cell concentrations of single-cell suspensions were counted using a hemocytometer  and adjusted to 1,000 cells/μl. Single-cell suspensions were loaded on the Chromium Single Cell Controller Instrument  to generate single cell gel beads in emulsions (GEMs), and 10×Genomics Chromium barcoding system was adopted to construct 10×barcoded cDNA library according to the manufacturer’s instructions. All libraries were sequenced on the Illumina HiSeq X Ten sequencing platform  with 150 bp pair-end module.https://github.com/alexdobin/STAR). Finally, the gene expression matrices of each cell were obtained for downstream visualization. After use of the CellRanger™ pipeline, a filter criterion was used to obtain high-quality cells. To remove multiple captured cells, the unique molecular identifier (UMI) count matrix , and the cells with UMI from 500 to 10,000 were selected. The double T-cells generated from two or more cells, were also removed using ‘DoubletFinder’ in Seurat. For each sample, we filtered the genes detected in fewer than three cells and cells with the total number of detected genes (nGenes) less than 500 and more than 3000. Following visual inspection of the distribution of cells by the fraction of mitochondrial genes expressed, low-quality cells where more than 25% of the counts belonged to mitochondrial genes, were further discarded.The CellRanger ™ pipeline (v3.0.0) was used to pre-process the raw sequencing data, including demultiplexing of cellular barcodes, evaluation of the total number of cells, and applying map reads to the genome. Briefly, after demultiplex cellular barcodes, the total number of cells captured in each sample were evaluated based on CellRanger ‘cell ranger count’ embedded function. The generated data were then mapped to the GRCh38.p5 human reference genome using the STAR aligner  and divided into three groups: significantly enriched genes , strictly enriched genes , and specific molecular marker genes of all cell groups. To sub-group cell clusters of interest for a more in-depth analysis, the ‘SubsetData’ function in Seurat was used to extract each cluster of interest. The extracted subclusters were also annotated with the previously reported maker genes to provide higher resolution for dissecting cellular heterogeneity among particular types of cells. Based on the significantly enriched genes for all samples, gene enrichment was conducted on the specific subclusters of each sample, and the GO and KEGG Pathways for the enriched gene were enriched using the ‘clusterProfiler’ software package.Based on the significantly enriched genes for each subcluster, cell differentiation trajectories were analyzed. Monocle (v 2.10.0) software  was usedp-value below 0.05 and of average log expression exceeding 0.1 were deemed significant. The interaction between cells was visualized using Cytoscape (Cell-cell interactions between different cell types were predicted based on known ligand–receptor pairs by Cellphone DB v2.1.0 . Permutaytoscape .The data presented in the study are deposited in the Sequence Read Archive (SRA) repository, accession number PRJNA847197.The studies involving human participants were reviewed and approved by Ethics Committee of Shanxi Bethune Hospital, and complied with all relevant ethical regulations (SBQLL-2020-038). The patients/participants provided their written informed consent to participate in this study. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.HC-Z and C-ZC designed and performed experiments, analyzed the data, and wrote the paper. H-QS, X-XW, and LZ performed the experiments and analyzed data. J-FH and H-LZ provided human samples. H-LZ and J-FH initiated and designed the study. J-FH initiated the study, organized, designed, and wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the Shanxi Science and Technology Department , Shanxi Province Human Resources and Social Security Department System (Grant No. 20210001), Research Project Supported by Shanxi Scholarship Council of China (Grant No. 2021-116), and Shanxi ‘136’ Leading Clinical Key Specialty (Grant No. 2019XY002). Shanxi Bethune Hospital provided some help in specimen collection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.